A combination of snps for tracing plasmodium vivax and application thereof

Abstract

The application discloses a Plasmodium vivax SNP molecular marker combination and application thereof in tracing. The 15 SNP molecular markers provided by the application are independent of each other, and the molecular marker combination can effectively identify Plasmodium vivax in Anhui, Henan and Zhejiang. The 15 SNP molecular markers are obtained by high-throughput gene mass spectrometry chip method, a Plasmodium vivax SNP information group is formed by the obtained effective SNP sites, and Plasmodium vivax tracing is further performed. The gene mass spectrometry chip method based on the SNP molecular marker is used for Plasmodium vivax DNA tracing, has the characteristics of high throughput and rapidness, and the 15 SNPs molecular markers are stably and widely present in Plasmodium vivax genomic DNA, and are helpful to large-scale popularization of the tracing technology.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection, specifically relating to a SNP combination for tracing the source of Plasmodium vivax and its application, and more specifically to a molecular identifier combination of Plasmodium vivax SNPs and its application in tracing the source of Plasmodium vivax in Anhui, Henan and Zhejiang provinces. Background Technology

[0002] Molecular source tracing technology is of significant value in determining the origin of Plasmodium infection. Traditional case-based epidemiological investigations are relatively subjective and lack sufficient evidence, while molecular source tracing technology utilizes geographically specific molecular markers combined with population genetic analysis to make a more objective determination. Among these, microsatellites (simple sequence repeats, SSRs) and single nucleotide polymorphisms (SNPs) are commonly used molecular markers in molecular source tracing research.

[0003] SSRs are tandem repeat sequences consisting of 2-6 nucleotides in the genome, also known as short tandem repeat sequences. They have high polymorphism and are easily amplified and detected by PCR.

[0004] SNPs are single nucleotide variations in the genome. First, they are distributed at a high density in the Plasmodium genome, with far more detectable sites than SSRs. Second, SNPs are highly stable and less prone to mutation. Third, there are various methods for SNP detection, including PCR amplification, sequencing, and microarray technology, making automation and high-throughput analysis easily achievable. Fourth, SNP detection has lower sample quality requirements than SSR detection. Amplified fragments can be smaller than 100 bp, and more information can be obtained from degraded DNA samples.

[0005] With the publication of the genome sequence of Plasmodium falciparum in 2002 and the complete genome sequence of Plasmodium nigra in 2012, a large number of SNP markers that may contain barcodes were discovered.

[0006] One limitation of these SNP combinations is the insufficient size of the datasets they are based on. As more available data becomes available and geographical coverage increases, researchers can develop more accurate and globally comprehensive genotyping tools. Furthermore, some SNP combinations used for tracing origins are analyzed and screened only at the bioinformatics level, without sufficient experimental samples to verify their accuracy. Moreover, some valuable samples that could significantly improve the accuracy of tracing origins have become difficult to use for testing due to improper long-term preservation.

[0007] Our team analyzed whole-genome data from 309 Plasmodium vivax parasites. Using comparative genomics methods, we screened for unique SNPs of Plasmodium vivax from Anhui, Henan, and Zhejiang provinces, and designed and evaluated corresponding gene mass spectrometry chips. We collected samples from 278 imported Plasmodium vivax cases from Anhui, Henan, Zhejiang, and Yunnan provinces. Using gene mass spectrometry chips, we identified molecular marker combinations that can effectively identify Plasmodium vivax from Anhui, Henan, and Zhejiang provinces, establishing a gene tracing technology that can objectively trace the origin of Plasmodium vivax, and developing products for Plasmodium vivax tracing.

[0008] Our team's approach is based on: 1) a large amount of whole-genome data: geographically extensive and containing 65 newly sequenced whole-genome datasets and 244 publicly available whole-genome datasets. This ensures a large number of high-quality candidate SNPs can be screened for bioinformatics analysis; 2) a large number of 278 vivax malaria case samples for identification. Furthermore, the application of gene mass spectrometry allows for the detection of even valuable but degraded DNA samples, significantly improving the accuracy of molecular marker identification; 3) the gene mass spectrometry method used can simultaneously perform 40-fold genotyping on up to 384 samples within 10 hours, generating 15,360 genotypes for analysis, achieving high-throughput analysis in a short time; and 4) the molecular marker combinations for tracing the origin of Plasmodium vivax obtained through screening and identification can be used for PCR testing at the grassroots level. Summary of the Invention

[0009] One of the technical problems this invention aims to solve is to provide a combination of SNP molecular markers for tracing the source of Plasmodium vivax in Anhui, Henan, and Zhejiang provinces. The 'n' in the sequence listing represents the mutation sites of each molecular marker, as follows:

[0010] SNP1: The sequence is shown in SEQ ID NO: 1, and the allele mutation site is: T / C;

[0011] SNP2: The sequence is shown in SEQ ID NO: 2, and the allele mutation site is: T / G;

[0012] SNP3: The sequence is shown in SEQ ID NO: 3, and the allele mutation site is: T / C;

[0013] SNP4: The sequence is shown in SEQ ID NO: 4, and the allele mutation site is: A / G;

[0014] SNP5: The sequence is shown in SEQ ID NO: 5, and the allele mutation site is: G / A;

[0015] SNP6: The sequence is shown in SEQ ID NO: 6, and the allele mutation site is C / T;

[0016] SNP7: The sequence is shown in SEQ ID NO: 7, and the allele mutation sites are: A / C;

[0017] SNP8: The sequence is shown in SEQ ID NO: 8, and the allele mutation site is C / T;

[0018] SNP9: The sequence is shown in SEQ ID NO: 9, and the allele mutation site is: C / T;

[0019] SNP10: The sequence is shown in SEQ ID NO: 10, and the allele mutation site is C / G;

[0020] SNP11: The sequence is shown in SEQ ID NO: 11, and the allele mutation site is: T / C;

[0021] SNP12: The sequence is shown in SEQ ID NO: 12, and the allele mutation site is C / T;

[0022] SNP13: The sequence is shown in SEQ ID NO: 13, and the allele mutation site is: G / T;

[0023] SNP14: The sequence is shown in SEQ ID NO: 14, and the allele mutation site is: G / T;

[0024] SNP15: The sequence is shown in SEQ ID NO: 15, and the allele mutation site is C / T.

[0025] The second technical problem to be solved by this invention is to provide a method for tracing the source of the Plasmodium vivax SNP molecular marker combination, comprising the following steps:

[0026] 1) Select 15 SNPs from the previous whole-genome sequencing data. The SNPs should meet the following requirements: A. The distance between any two adjacent SNPs is >1 Mb, ensuring that the SNPs are independent and there is no linkage; B. No association between any SNP and other SNPs; C. No variation in the flanking sequences of the SNPs.

[0027] 2) Collect 128 whole blood samples from patients with Plasmodium vivax from Anhui, Henan and Zhejiang provinces and 150 samples from Yunnan province, and extract their DNA;

[0028] 3) Based on the selected 15 SNP sites, specific primers and probes were designed, and the SNP site genotyping of each sample was performed using the gene mass spectrometry chip method, including: gene amplification, PCR product purification, extension reaction, desalting, chip spotting, and genotyping.

[0029] The third technical problem to be solved by this invention is to provide specific primers for the use of the Plasmodium vivax SNP molecular marker combination in the gene mass spectrometry chip method, namely:

[0030] SNP1: Upstream primer snp1-F is shown in SEQ ID NO: 16; Downstream primer snp1-R is shown in SEQ ID NO: 17;

[0031] SNP2: Upstream primer snp2-F is shown in SEQ ID NO: 18; Downstream primer snp2-R is shown in SEQ ID NO: 19;

[0032] SNP3: Upstream primer snp3-F is shown in SEQ ID NO: 20; Downstream primer snp3-R is shown in SEQ ID NO: 21;

[0033] SNP4: Upstream primer snp4-F is shown in SEQ ID NO: 22; Downstream primer snp4-R is shown in SEQ ID NO: 23;

[0034] SNP5: Upstream primer snp5-F is shown in SEQ ID NO: 24; Downstream primer snp5-R is shown in SEQ ID NO: 25;

[0035] SNP6: Upstream primer snp6-F is shown in SEQ ID NO: 26; Downstream primer snp6-R is shown in SEQ ID NO: 27;

[0036] SNP7: Upstream primer snp7-F is shown in SEQ ID NO: 28; Downstream primer snp7-R is shown in SEQ ID NO: 29;

[0037] SNP8: Upstream primer snp8-F is shown in SEQ ID NO: 30; Downstream primer snp8-R is shown in SEQ ID NO: 31;

[0038] SNP9: Upstream primer snp9-F is shown in SEQ ID NO: 32; Downstream primer snp9-R is shown in SEQ ID NO: 33;

[0039] SNP10: Upstream primer snp10-F is shown in SEQ ID NO: 34; Downstream primer snp10-R is shown in SEQ ID NO: 35;

[0040] SNP11: Upstream primer snp11-F is shown in SEQ ID NO: 36; Downstream primer snp11-R is shown in SEQ ID NO: 37;

[0041] SNP12: Upstream primer snp12-F is shown in SEQ ID NO: 38; Downstream primer snp12-R is shown in SEQ ID NO: 39;

[0042] SNP13: Upstream primer snp13-F is shown in SEQ ID NO: 40; Downstream primer snp13-R is shown in SEQ ID NO: 41;

[0043] SNP14: Upstream primer snp14-F is shown in SEQ ID NO: 42; Downstream primer snp14-R is shown in SEQ ID NO: 43;

[0044] SNP15: Upstream primer snp15-F is shown in SEQ ID NO: 44; downstream primer snp15-R is shown in SEQ ID NO: 45.

[0045] The fourth technical problem to be solved by this invention is to provide specific probes for the use of the Plasmodium vivax SNP molecular marker combination in the gene mass spectrometry chip method, namely:

[0046] SNP1: Probe snp1-E is shown in SEQ ID NO: 46;

[0047] SNP2: Probe snp2-E is shown in SEQ ID NO: 47;

[0048] SNP3: Probe snp3-E is shown in SEQ ID NO: 48;

[0049] SNP4: Probe snp4-E is shown in SEQ ID NO: 49;

[0050] SNP5: Probe snp5-E as shown in SEQ ID NO: 50.

[0051] SNP6: Probe snp6-E is shown in SEQ ID NO: 51;

[0052] SNP7: Probe snp7-E is shown in SEQ ID NO: 52;

[0053] SNP8: Probe snp8-E is shown in SEQ ID NO: 53;

[0054] SNP9: Probe snp9-E is shown in SEQ ID NO: 54;

[0055] SNP10: Probe snp10-E as shown in SEQ ID NO: 55.

[0056] SNP11: Probe snp11-E is shown in SEQ ID NO: 56;

[0057] SNP12: Probe snp12-E is shown in SEQ ID NO: 57;

[0058] SNP13: Probe snp13-E is shown in SEQ ID NO: 58;

[0059] SNP14: Probe snp14-E is shown in SEQ ID NO: 59;

[0060] SNP15: Probe snp15-E as shown in SEQ ID NO: 60.

[0061] The fifth technical problem to be solved by this invention is the application of the aforementioned SNP molecular marker combination, specific primers, and probes in the tracing of Plasmodium vivax in Anhui, Henan, and Zhejiang provinces, or in the preparation of products for tracing Plasmodium vivax in Anhui, Henan, and Zhejiang provinces.

[0062] Beneficial effects

[0063] This invention is based on previous whole-genome resequencing work. Using comparative genomics methods, specific SNPs of Plasmodium vivax from Anhui, Henan, and Zhejiang provinces were selected. Corresponding gene mass spectrometry chips were designed and evaluated. Molecular marker combinations that can effectively identify Plasmodium vivax from Anhui, Henan, and Zhejiang provinces were screened and identified. These SNP combinations can be used to develop products for tracing the origin of Plasmodium vivax, accurately assessing the transmission risk of epidemic sites, and thus enabling timely and effective control measures.

[0064] This invention has the following advantages: 1) It is based on a large amount of whole-genome data: wide geographical coverage and large data volume. This ensures that a large number of high-quality candidate SNPs can be screened at the bioinformatics analysis level; 2) It is based on a large number of vivax malaria case samples used for identification, and some precious but degraded DNA samples can also be used for detection, which greatly improves the accuracy of molecular marker identification; 3) The gene mass spectrometry chip method used can generate up to 15,360 genotypes for analysis within 10 hours, achieving high-throughput analysis in a short time; 4) The molecular marker combinations for tracing the source of Plasmodium vivax obtained through screening and identification can be used for PCR experiments at the grassroots level. Attached Figure Description

[0065] Figure 1 This is a schematic diagram of the genotyping results of Plasmodium vivax specific SNP sites using the gene mass spectrometry chip method in Example 1. Detailed Implementation

[0066] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0067] Example 1

[0068] 1. Materials

[0069] 1.1 Genomic DNA of Plasmodium vivax

[0070] Whole blood samples were collected from 278 patients infected with Plasmodium vivax from Henan, Anhui, Zhejiang and Yunnan provinces.

[0071] 1.2 Main Reagents

[0072]

[0073] 2 Methods

[0074] 2.1 Selection of SNP sites

[0075] SNP sites were selected from previous research results to ensure that: 1) the distance between any two adjacent SNPs is ≥1 Mb, and the SNPs are independent of each other and there is no linkage; 2) any SNP site is not associated with other molecular markers; and 3) there are no variations in the flanking sequences of the SNPs. The SNP sites selected in this study are shown in Table 1 (SEQ ID NO: 1-15).

[0076] Table 1. Information related to SNP sites

[0077]

[0078] 2.2 Amplification of SNP molecular markers in Plasmodium vivax

[0079] Based on the upstream and downstream sequence information of the selected SNPs, 15 pairs of specific primers were designed on the Agena company's primer design website (https: / / www.agenacx.com). The primer information used in this invention is shown in Table 2, and the primers were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0080] Table 2. Primer information for amplification of Plasmodium vivax SNPs (SEQ ID NO: 16-45)

[0081]

[0082] Gene amplification was performed using Plasmodium vivax genomic DNA as a template. Reaction conditions.

[0083] The reaction mixture consisted of: ① 95 °C pre-denaturation for 2.0 min; ② 95 °C denaturation for 30 sec; ③ 56 °C annealing for 30 sec; ④ 72 °C extension for 1.0 min, for 45 cycles; ⑤ 72 °C extension for 5 min, and finally stored at 4 °C. DNA polymerase was purchased from Agena Bioscience. The reaction volume was 5.0 μl, specifically: 0.5 μl of 10 x PCR Buffer; 0.4 μl of MgCl2; 0.1 μl of dNTPs (2.5 mM each); 1 μl of 0.5 μM Primer Mix; 2 μl of Template DNA; 0.2 μl of PCR Enzyme (5 U / μl); and 0.8 μl of HPLC-grade water.

[0084] 2.3 Purification of PCR products

[0085] The PCR products were purified using the SAP method. The reaction conditions were: ① 37 ℃ for 40.0 min, ② 85 ℃ for 5.0 min, and finally stored at 4 ℃. The SAP purification enzyme was purchased from Agena Bioscience. The reaction volume was 7.0 μl, specifically: 0.17 μl SAP Buffer; 0.3 μl SAP Enzyme (1.7 U / μl); 5 μl PCR product; and 1.53 μl ultrapure water.

[0086] 2.4 Extension reaction

[0087] Based on the upstream and downstream sequence information of the selected SNPs, 15 specific probes were designed. The probe information used in this invention is shown in Table 3. They were synthesized by Nanjing GenScript Co., Ltd. (SEQ ID NO: 46-60).

[0088]

[0089] The purified PCR product was used as a template for extension reactions. The reaction conditions were: ① 95 °C pre-denaturation for 30 sec; ② 95 °C denaturation for 5 sec; ③ 52 °C annealing for 5 sec; ④ 80 °C extension for 5 sec; ③ to ④, 5 cycles; ② to ④, 40 cycles; ⑤ 72 °C extension for 3 min, and finally stored at 4 °C. The extension enzyme was purchased from Agena Bioscience. The reaction volume was 9.0 μl, specifically: 0.2 μl iPLEX Buffer; 0.2 μl iPLEX Termination mix; 0.94 μl Extend Mix (5 probes); 0.04 μl iPLEX Enzyme; 7 μl purified PCR product; and 0.04 μl Water.

[0090] 2.5 Sample desalination

[0091] Using a spatula, evenly distribute the cleaning resin into the 96 wells of the PCR plate. Add 41 μl of water to the extension product, cover with a sealing film, and centrifuge to mix. Invert the 96-well PCR plate containing the extension product onto the plate containing the cleaning resin, invert the PCR plate again, cover with a sealing film, and place on a vortex mixer to mix thoroughly for 15 minutes. Centrifuge the PCR plate at 4000 rpm for 5 minutes.

[0092] 2.6 Genotyping and Statistical Analysis

[0093] The desalted extension products from the PCR plate were spotted onto a gene chip, which was then placed on an Agena MassArray mass spectrometer for genotyping. The mass spectrometry results were statistically analyzed using Agena's genotyping software version 4.0.

[0094] 3 Results

[0095] 3.1 Genotyping

[0096] Genotyping of SNPs was performed on 278 Malaria vivax samples using Agena's MassArray mass spectrometer, and the allele distribution of each SNP was calculated. The main genotypes of samples from Anhui, Henan, and Zhejiang provinces (Sample A) and Yunnan province (Sample B) are as follows:

[0097]

[0098] 3.2 Analysis of the detection rate of Plasmodium vivax in samples

[0099] According to statistics, among 278 Plasmodium vivax samples, 271 samples showed signals in the gene mass spectrometry chip results, with a detection rate of 97.5%.

[0100] 3.3 Sensitivity and Specificity Analysis

[0101] In the results of the detected signals, based on the combination of 15 SNPs, 121 out of 125 Plasmodium vivax samples from Anhui, Henan and Zhejiang provinces were identified as originating from Anhui, Henan and Zhejiang provinces, with a sensitivity of 96.8%; and 12 out of 146 Plasmodium vivax samples from Yunnan province were identified as originating from Anhui, Henan and Zhejiang provinces, with a specificity of 91.8%.

[0102]

[0103]

[0104] The results above show that our team has collected samples of imported vivax malaria cases from Anhui, Henan, Zhejiang, and Yunnan provinces in recent years, and performed whole-genome resequencing on some Plasmodium vivax samples. Using comparative genomics methods, we selected unique SNPs for Plasmodium vivax from Anhui, Henan, and Zhejiang provinces, designed and evaluated corresponding gene mass spectrometry chips, and screened and identified 15 SNP molecular marker combinations that can effectively identify Plasmodium vivax from Anhui, Henan, and Zhejiang provinces. These SNP combinations achieved a sensitivity of 96.8% and a specificity of 91.8%. Based on this invention, our team has successfully established a gene tracing technology that can objectively trace the origin of vivax malaria. These achievements can be used for malaria case tracing after malaria elimination in China, and can also be applied to the development of Plasmodium vivax tracing products, showing broad application prospects.

[0105] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A molecular marker combination of SNPs for tracing the origin of Plasmodium vivax, characterized in that: SNPs are molecular identifiers: SNP1: The sequence is shown in SEQ ID NO: 1, and the allele mutation site is: T / C; SNP2: The sequence is shown in SEQ ID NO: 2, and the allele mutation site is: T / G; SNP3: The sequence is shown in SEQ ID NO: 3, and the allele mutation site is: T / C; SNP4: The sequence is shown in SEQ ID NO: 4, and the allele mutation site is: A / G; SNP5: The sequence is shown in SEQ ID NO: 5, and the allele mutation site is: G / A; SNP6: The sequence is shown in SEQ ID NO: 6, and the allele mutation site is C / T; SNP7: The sequence is shown in SEQ ID NO: 7, and the allele mutation sites are: A / C; SNP8: The sequence is shown in SEQ ID NO: 8, and the allele mutation site is C / T; SNP9: The sequence is shown in SEQ ID NO: 9, and the allele mutation site is: C / T; SNP10: The sequence is shown in SEQ ID NO: 10, and the allele mutation site is C / G; SNP11: The sequence is shown in SEQ ID NO: 11, and the allele mutation site is: T / C; SNP12: The sequence is shown in SEQ ID NO: 12, and the allele mutation site is C / T; SNP13: The sequence is shown in SEQ ID NO: 13, and the allele mutation site is: G / T; SNP14: The sequence is shown in SEQ ID NO: 14, and the allele mutation site is: G / T; SNP15: The sequence is shown in SEQ ID NO: 15, and the allele mutation site is C / T. The source is identified as Plasmodium vivax from (1) Anhui Province, Henan Province and Zhejiang Province; or (2) Yunnan Province.

2. The application of a reagent for detecting the SNP molecular marker combination genotype as described in claim 1 in the tracing of Plasmodium vivax or in the preparation of products for tracing the origin of Plasmodium vivax, wherein the tracing is for identifying Plasmodium vivax as originating from (1) Anhui Province, Henan Province and Zhejiang Province; or (2) Yunnan Province.

3. A primer pair and probe for detecting the SNP molecular marker combination genotype as described in claim 1 in the tracing of Plasmodium vivax or in the preparation of products for tracing the origin of Plasmodium vivax, wherein the tracing is for identifying Plasmodium vivax as originating from (1) Anhui Province, Henan Province, and Zhejiang Province; or (2) Yunnan Province, The primer pair is: SNP1: Upstream primer snp1-F is shown in SEQ ID NO: 16; Downstream primer snp1-R is shown in SEQ ID NO: 17; SNP2: Upstream primer snp2-F is shown in SEQ ID NO: 18; Downstream primer snp2-R is shown in SEQ ID NO: 19; SNP3: Upstream primer snp3-F is shown in SEQ ID NO: 20; Downstream primer snp3-R is shown in SEQ ID NO: 21; SNP4: Upstream primer snp4-F is shown in SEQ ID NO: 22; Downstream primer snp4-R is shown in SEQ ID NO: 23; SNP5: Upstream primer snp5-F is shown in SEQ ID NO: 24; Downstream primer snp5-R is shown in SEQ ID NO: 25; SNP6: Upstream primer snp6-F is shown in SEQ ID NO: 26; Downstream primer snp6-R is shown in SEQ ID NO: 27; SNP7: Upstream primer snp7-F is shown in SEQ ID NO: 28; Downstream primer snp7-R is shown in SEQ ID NO: 29; SNP8: Upstream primer snp8-F is shown in SEQ ID NO: 30; Downstream primer snp8-R is shown in SEQ ID NO: 31; SNP9: Upstream primer snp9-F is shown in SEQ ID NO: 32; Downstream primer snp9-R is shown in SEQ ID NO: 33; SNP10: Upstream primer snp10-F is shown in SEQ ID NO: 34; Downstream primer snp10-R is shown in SEQ ID NO: 35; SNP11: Upstream primer snp11-F is shown in SEQ ID NO: 36; Downstream primer snp11-R is shown in SEQ ID NO: 37; SNP12: Upstream primer snp12-F is shown in SEQ ID NO: 38; Downstream primer snp12-R is shown in SEQ ID NO: 39; SNP13: Upstream primer snp13-F is shown in SEQ ID NO: 40; Downstream primer snp13-R is shown in SEQ ID NO: 41; SNP14: Upstream primer snp14-F is shown in SEQ ID NO: 42; Downstream primer snp14-R is shown in SEQ ID NO: 43; SNP15: Upstream primer snp15-F is shown in SEQ ID NO: 44; Downstream primer snp15-R is shown in SEQ ID NO: 45; The probe is: SNP1: Probe snp1-E is shown in SEQ ID NO: 46; SNP2: Probe snp2-E is shown in SEQ ID NO: 47; SNP3: Probe snp3-E is shown in SEQ ID NO: 48; SNP4: Probe snp4-E is shown in SEQ ID NO: 49; SNP5: Probe snp5-E is shown in SEQ ID NO: 50; SNP6: Probe snp6-E is shown in SEQ ID NO: 51; SNP7: Probe snp7-E is shown in SEQ ID NO: 52; SNP8: Probe snp8-E is shown in SEQ ID NO: 53; SNP9: Probe snp9-E is shown in SEQ ID NO: 54; SNP10: Probe snp10-E as shown in SEQ ID NO: 55; SNP11: Probe snp11-E is shown in SEQ ID NO: 56; SNP12: Probe snp12-E is shown in SEQ ID NO: 57; SNP13: Probe snp13-E is shown in SEQ ID NO: 58; SNP14: Probe snp14-E is shown in SEQ ID NO: 59; SNP15: Probe snp15-E as shown in SEQ ID NO: 60.

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